The HIV accessory protein Vpu is known to modulate two host proteins, CD4 and BST-2, but it is thought to do so by two completely different mechanisms. CD4 is believed to be targeted in the endoplasmic reticulum for proteasomal degradation, while BST-2 is believed to be targeted in the Golgi apparatus and the recycling endosomes for lysosomal degradation. Both targets are believed to interact with cellular Skp-Cullin1-F-box (SCF) E3 ligases to facilitate their action, but it is disputed which kinds of SCF ligase act as co-factors. SCF ligases that contain the F- box proteins ssTRCP-1 or -2 are believed to be two potential Vpu cofactors, but some suggest that there are additional forms of the ligase that have not yet been identified. Because different assays are typically used to study these two Vpu targets, it has been difficult to draw direct parallels between them. In particular, the assays used to study CD4 are performed under conditions where Vpu is expressed at levels much higher than would be found in a normal infection. The glycoprotein from Gibbon ape leukemia virus (GaLV Env) has recently been identified as an additional Vpu target. Based on recognition parameters, GaLV Env appears to be analogous to CD4, but based on its mechanism of action, it appears more analogous to BST-2. Vpu requires an SCF ligase to target GaLV Env, but ssTRCP-1 and -2 are dispensable. In addition, CD4 expression was found to restrict HIV-1 infectivity under particular conditions in a BST-2 and HIV Env independent fashion, but this restriction could be alleviated by Vpu. A novel assay has been developed based on this observation for studying Vpu targeting of CD4 at physiological concentrations. It is hypothesized that CD4, BST-2, and GaLV Env are all modulated by a single mechanism and that there is a previously unidentified F-box protein that plays a critical role in this modulation An infectivity-based assay will be used to identify the SCF ligase used by Vpu to target GaLV Env, and characterize the importance of this ligase in the modulation of all three Vpu targets. The work will be divided into the following specific aims: Aim 1. To determine how CD4 restricts viral infectivity in the absence of HIV-1 Env and how Vpu overcomes this restriction. Aim 2. To identify the F-box protein used by Vpu to modulate GaLV Env and to determine if this F-box protein also functions in the modulation of CD4 and BST-2. These studies will answer fundamental questions about the mechanism of HIV-1 Vpu activity and could open up new avenues for the development of therapeutic antivirals.